Recombinant allergen with reduced ige binding but undiminished t-cell antigenicity

ABSTRACT

The present invention relates generally to reagents useful in the immunotherapeutic or immunoprophylactic treatment of allergic diseases. More particularly, the present invention provides modified allergens exhibiting reduced IgE interactivity including reduced IgE production-stimulatory activity, while retaining T-cell antigenicity, which are useful in the immunomodulation of type I allergic disease conditions. The present invention further contemplates a method of immunomodulation of allergic diseases such as type I allergic disease conditions by the administration of modified allergens exhibiting reduced IgE interactivity while retaining T-cell antigenicity.

FIELD OF THE INVENTION

The present invention relates generally to reagents useful in theimmunotherapeutic or immunoprophylactic treatment of allergic diseases.More particularly, the present invention provides modified allergensexhibiting reduced IgE interactivity including reduced IgEproduction-stimulatory activity, while retaining T-cell antigenicity,which are useful in the immunomodulation of type I allergic diseaseconditions. The present invention further contemplates a method ofimmunomodulation of allergic diseases such as type I allergic diseaseconditions by the administration of modified allergens exhibitingreduced IgE interactivity while retaining T-cell antigenicity.

BACKGROUND OF THE INVENTION

Bibliographic details of references provided in the subjectspecification are listed at the end of the specification.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Type I allergic diseases such as seasonal allergic rhinitis (hayfever),conjunctivitis, allergic asthma and allergic dermatitis represent amajor health problem in industrialized countries (Wuthrich, Int. Arch.Allergy Immunol. 90: 3-10, 1989). It is currently estimated that 15-20%of the population in developed countries are afflicted with some form ofallergy (Miyamoto, Advances in Allergology and Clinical Immunology.Godard P, Bousquet J, Michel FB (eds) pp. 343-347. The ParthenonPublishing Group, Cornforth, UK, 1992). Therefore, the diagnosis andtherapy of these diseases have become focal points of interest forscientific investigation.

The primary immunological and biochemical bases of type I allergicreactions are the interaction of allergenic substances (allergens) withIgE antibodies bound to high affinity Fc receptors on the surface ofmast cells and basophils. This interaction results in cross linking ofallergen-specific IgE antibodies which in turn stimulates an immediaterelease and cascade production of inflammatory mediators responsible forallergic symptoms. Allergens are present in airborne particles such ashouse-dust, pollen of grasses, weeds and trees, mould spores and animaldander.

At present, one form of therapeutic intervention of allergic diseases(such as rhinitis, conjunctivitis and allergic asthma) involvesinjection of the allergen assumed to be responsible for the allergicresponse. This is referred to as hypo-sensitization treatment. Extractscurrently in use in this procedure are prepared from natural sources andcontain, in addition to the allergens, components such as proteins towhich patients are not allergic.

The development of recombinant techniques has provided the means toproduce high levels of purified allergens for diagnostic and therapeuticpurposes. However, the high level of purity of recombinant allergenpreparations results in a high anaphylactogenic index even at very lowdoses. Accordingly, extreme care is required when they are administeredto patients. There is a need, therefore, to develop recombinantallergens with a reduced risk of anaphylactic shock.

The major outdoor cause of seasonal hay-fever and allergic asthma isairborne grass pollen (Smart et al., Int. Arch. Allergy Immunol. 7:243-248, 1983). Pollen calenders show that grass pollen is most abundantin spring and early summer when grasses flower and this is when allergicasthma peaks in incidence. The most important sources of grass pollenare common agricultural pasture grasses which have been widelyintroduced throughout the world, but vary in temperature and tropicalclimate zones. In cool temperature regions, grasses such as rye-grass,Kentucky bluegrass and timothy (all belonging to the subfamily Pooideae)are of clinical significance, whereas in warm temperature andsubtropical environments pollen of Bermuda grass (subfamilyChloridoideae) becomes the most important source of allergens. The mostcomprehensive studies have been made on proteins from rye-grass pollenand to a lesser extent Kentucky bluegrass and timothy.

Individuals sensitive to allergens from one grass are often sensitive tothose of a number of other grass genera. This is particularly true forpollen of grasses within the subfamily Pooideae (Smith et al., “Analysisof rye-grass pollen allergens using two dimensional electrophoresis andimmunoblotting.” In Kraft D (ed), Molecular Biology and Immunology ofAllergens, CRC Press, Boca Raton, Fla., 1994), where immunologicalcross-reactivity has been demonstrated in inhibition experiments usingan IgE-binding assay, the radioallergosorbent test (RAST). In theseexperiments, pollen extracts from one grass were able to inhibit bindingof IgE to extracts from other grasses.

Allergenic components of grass pollen can be classified into differentgroups according to their physiochemical and immunological properties.The major allergens that elicit an allergic reaction to pollen from thePooid grasses are group 1 and 5 allergens, as judged by both criteria ofthe number of allergic patients responding and relative amounts of IgEbinding to the allergens (Singh et al, 1991, supra). In case ofperennial rye-grass, Lolium perenne, pollen extracts contain more than17 proteins which have the capacity to bind IgE from sera of grasspollen allergic patients (Smith et al., 1993, supra). However, it hasbeen shown that allergens of group 1 and 5 together can inhibit most ofthe IgE binding to crude pollen extracts (Bond et al., J. Allergy Clin.Immunol. 91: 339, 1993).

Lol p 5, a protein of 28-33 kDa, is the second most prevalent rye-grassallergen which causes allergy in 85-90% of grass pollen allergicindividuals. Molecular cloning of cDNAs encoding this group 5 allergen(Singh et al., Proc. Natl. Acad. Sci. USA 88: 1384-1388,1991; Ong etal., Gene 134: 235-240, 1993) has shown that Lol p 5 exists as a familyof homologous but distinguishable isoforms which retain their IgEreactivity even after separation on denaturing SDS-PAGE gels andimmunoblotting (Singh et al., 1991, Supra).

A need, accordingly, exists to develop modified forms of recombinantallergens useful in immunotherapy and immunoprophylaxis of allergicconditions.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO: 1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

The present invention provides a modified recombinant allergen, whereinin naturally occurring form, the allergen is associated with allergicdisease conditions in sensitive subjects. Conveniently, the modifiedrecombinant allergen comprises an amino acid sequence modified from thenaturally occurring amino acid sequence such that the allergen lacks orcomprises reduced numbers of IgE epitopes and/or exhibits reducedbinding capacity for IgE and/or exhibits reduced IgEproduction-stimulatory activity while retaining T-cell antigenicity.

Preferably, the allergic disease condition is a type I allergic diseasecondition.

Preferably, the recombinant allergen is a grass pollen allergen.

Most preferably, the grass allergen is a rye-grass pollen allergen suchas but not limited to Lol p 5 or immunologically or botanically relatedallergens such as Ph1 p 5 and Poa p 5.

In a particularly preferred embodiment, the present invention provides amodified Lol p 5 allergen which lacks or comprises a reduced number ofIgE epitopes and/or exhibits reduced IgE binding capacity and/orexhibits reduced IgE production-stimulatory activity while retainingT-cell antigenicity wherein said Lol p 5 variant is selected from amolecule having the amino acid sequence set forth in SEQ ID NOS:8 to 12(see Table 1) or a modified allergen corresponding to an immunologicallyor botanically related allergen.

The present invention is further directed to a composition comprising amodified allergen such as a grass allergen (e.g. a rye-grass pollenallergen) which lacks or comprises reduced numbers of IgE epitopesand/or exhibits reduced binding capacity for IgE and/or exhibits reducedIgE production-stimulatory activity while retaining T-cell antigenicity.The composition further comprises one or more pharmaceuticallyacceptable carriers and/or diluents.

The present invention further contemplates a method for the prophylaxisor treatment of an allergic disease condition in a subject byadministering to the subject, an effective amount of a modified allergenwhich lacks or comprises reduced numbers of IgE epitopes and/or exhibitsreduced binding capacity for IgE and/or exhibits reduced IgEproduction-stimulatory activity while retaining T-cell antigenicity.

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 1. TABLE 1 Summary of sequenceidentifiers SEQUENCE ID NO: DESCRIPTION 1 Amino acid sequence of isoformA of Lol p 5 2 Amino acid sequence of isoform B of Lol p 5 3 Amino acidsequence of isoform A of Phl p 5 4 Amino acid sequence of isoform B ofPhl p 5 5 Amino acid sequence of isoform Poa p5 6 Amino acid sequence ofisoform Poa p5 7 Amino acid sequence of Lol p 5 variant 8 Amino acidsequence of Lol p 5 variant D1 9 Amino acid sequence of Lol p 5 variantD2 10 Amino acid sequence of Lol p 5 variant D3 11 Amino acid sequenceof Lol p 5 variant D4 12 Amino acid sequence of Lol p 5 variant D5 13Nucleotide sequence of forward primer used to clone out D1 14 Nucleotidesequence of reverse primer used to clone out D1 15 Nucleotide sequenceof forward primer used to clone out D2 16 Nucleotide sequence of reverseprimer used to clone out D2 17 Nucleotide sequence of forward primerused to clone out D3 18 Nucleotide sequence of reverse primer used toclone out D3 19 Nucleotide sequence of forward primer used to clone outD4 20 Nucleotide sequence of reverse primer used to clone out D4 21Nucleotide sequence of forward primer used to clone out D5 22 Nucleotidesequence of reverse primer used to clone out D5

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation showing a comparison of deduced amino acidsequences of group 5 allergens. Dashes indicate gaps which have beenintroduced to give maximum alignment. Residues identical to Lol p 5 Aare indicated by asterisks.

FIG. 2 is a representation showing amino acid sequence of Lol p 5 Aindicating the mutations introduced in the allergen to give D1, D2, D3,D4 and D5 mutants. Amino acids of Lol p 5 A that were changed areindicated by boxes, whereas new sequences are given in bold.

FIG. 3 is a schematic representation of mutated Lol p 5 variants; e.g.mut 1 contains mutation D1.

FIG. 4 is a representation showing sequences of primers used to createmutations in Lol p 5 A.

FIG. 5 is a diagrammatic representation showing slot blot analysis ofLol p 5 (non-mutated) and the nine mutated variants (mut 1 to mut 9) ofreactivities of the purified proteins to a polyclonal (p), a monoclonal(m) antibody and to sera of 7 rye-grass pollen-allergic patients.

FIG. 6 is a diagrammatic representation showing immunoblot analyses ofLol p 5 (non-mutated) and the nine mutated variants (mut 1 to mut 9) ofreactivities of the purified proteins to a polyclonal (p), a monoclonal(m) antibody and to the serum of a rye-grass pollen-allergic patient(patient 2).

FIGS. 7A, B and C are graphical representations of ELISA assays usingpurified non-mutated Lol p 5 and four of the mutated proteins (mut 3,mut 4, mut 6, mut 9) showing a reduction in reactivity of the mutatedvariants to a monoclonal (mAb A7) and to IgE of two patients (patient 4,patient 27).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, genetically engineeredsubstantially hypoallergenic, variants of allergens with an inability orreduced capacity to interact with IgE are provided for use inimmunotherapy and immunoprophylaxis. Certain types of modifications ofthe amino acid sequence are determined to result in a lack of or reducednumbers of IgE epitopes, reduced activity of IgE epitopes, reducedability to interact with IgE and/or reduced IgE production-stimulatoryactivity.

Accordingly, one aspect of the present invention provides a modifiedrecombinant allergen, wherein said allergen in naturally occurring formis associated with allergic disease conditions in sensitive subjects,wherein said modified recombinant allergen comprises an amino acidsequence modified from the naturally occurring amino acid sequence suchthat the allergen lacks or comprises reduced numbers of IgE epitopesand/or exhibits reduced binding capacity for IgE and/or exhibits reducedIgE production-stimulatory activity while retaining T-cell antigenicity.

The term “sensitive” subject is used in its broadest sense to include anindividual exhibiting the symptoms of an allergic disease and moreparticularly a type I allergic disease in response to or associated withthe allergen. An “individual” is preferably a human but also extends toa non-human primate, livestock animal (e.g. sheep, cow, pig, horse,donkey, goat), laboratory test animal (e.g. mouse, rat, rabbit, guineapig) and a companion animal (e.g. dog, cat).

The present invention is particularly directed to grass pollenallergens.

Accordingly, another aspect of the present invention is directed to amodified recombinant grass pollen allergen, wherein said grass pollenallergen in naturally occurring form is associated with type I allergicdisease-conditions wherein said modified recombinant grass pollenallergen comprises an amino acid sequence modified from the naturallyoccurring amino acid sequence such that the allergen lacks and/orcomprises reduced numbers of IgE epitopes or exhibits reduced bindingcapacity for IgE and/or exhibits reduced IgE production-stimulatoryactivity while retaining T-cell antigenicity.

In a particularly preferred embodiment, the grass pollen allergen is arye-grass or immunologically related grass pollen allergen such as butnot limited to Lol p 5, Ph1 p 5 and Pao p 5. Reference to “grass pollenallergen” includes all rye-grass or immunologically related grass pollenallergens or other grass allergens.

Accordingly, this aspect of the present invention contemplates amodified recombinant rye grass pollen allergen comprising an amino acidsequence modified from the naturally occurring amino acid sequence suchthat the allergen lacks or comprises reduced numbers of IgE epitopesand/or exhibits reduced binding capacity for IgE and/or exhibits reducedIgE production-stimulatory activity while retaining T-cell antigenicity.

The retention of T-cell antigenicity includes reference to the retentionof T-cell epitopes or an otherwise capacity to interact with T cells toelicit a T-cell response.

The present invention is hereinafter described in relation to Lol p 5.This is done since Lol p 5, up to the present time, represents aparticularly useful allergen in which to practice the present invention.This is done, however, with the understanding that the present inventionextends to any allergen, especially an allergen involved in type Iallergic diseases such as but not limited to a group 5 grass pollenallergen. The methods of the present invention are particularlyapplicable to any rye-grass or immunologically related grass pollenallergen in addition to Lol p 5 such as Ph1 p 5 and Poa p 5.

In work leading up to the present invention, the inventors expressedrecombinant Lol p 5 in E. coli as a nonfusion protein and found thatremoval of the N-terminal signal peptide from the cDNA prior to cloninginto the bacterial expression vector resulted in a soluble recombinantform of the allergen. This approach made it possible to avoid the harshdenaturing conditions for isolation of the allergen from bacterialcells. Recombinant Lol p 5 was tested for antigenic similarity with itsnatural counterpart by inhibition ELISA experiments and the inventorsshowed that the recombinant form fully inhibited IgE binding of anisolated form of its natural pollen counterpart. The fact that singlerecombinant isoforms can inhibit IgE binding to natural allergensfurther implied that different allergen isoforms were similar. Theinventors used recombinant allergens in immunoblot inhibition studieswhere allergic sera reincubated with recombinant Lol p 5 were used toprobe two dimensional immunoblots of rye-grass soluble protein. It wasfound in accordance with the present invention that preincubation withone form completely abolished binding to all the different forms encodedby different genes. This showed that even with sequencemicro-heterogenities, different allergen isoforms were antigenicallyvery similar.

The next-step in the development of the present invention was todetermine key amino acid residues of the allergenic proteins which couldbe changed which removed or reduced IgE interactivity while maintainingthe general structure and functionality of the T-cell epitopes.

The inventors determined which amino acid residues on isoforms A and Bof Lol p 5 were conserved. It was reasoned that such conserved aminoacid residues would be important for IgE binding since there iscross-reactivity between a number of allergens from different grasses.By selectively mutating these conserved amino acid residues, mutantswere identified which have no or reduced IgE interactivity whileretaining T-cell antigenicity.

Accordingly, another aspect of the present invention comprises amodified group 5 grass pollen allergen, wherein said group 5 grasspollen allergen comprises a substitution, deletion and/or addition atone or more amino acid residues which is/are conserved in at least twoimmunologically cross-reactive group 5 grass pollen allergens andwherein said modified group 5 grass pollen allergen lacks or comprisesreduced numbers of IgE epitopes and/or exhibits reduced binding capacityfor IgE and/or exhibits reduced IgE production-stimulatory activitycompared to the corresponding naturally occurring form.

In accordance with the aspect of the present invention, one suitablereference amino acid sequence is SEQ ID NO:1 which is the amino acidsequence of Lol p 5, isoform A. An amino acid sequence comparison, suchas in FIG. 1, shows the conserved amino acid residues in isoforms A andB of Lol p 5 and Ph1 p 5 and in isoforms of Poa p 5. Conserved residuesin FIG. 1 are indicated by asterisks. Mutants are then readilyintroduced which alter one or more of these conserved residues.

Another aspect of the present invention provides a modified group 5grass pollen allergen comprising an amino acid truncation orsubstitution, deletion and/or addition at a position corresponding toone or more of mutants 1 to 9 of Lol p 5 as depicted in FIG. 3 or acorresponding mutant in an immunologically related allergen.

In a particularly preferred embodiment, the present invention provides amodified Lol p 5 allergen which lacks or comprises a reduced number ofIgE epitopes and/or exhibits reduced IgE binding capacity and/orexhibits reduced IgE production-stimulatory activity while retainingT-cell antigenicity wherein said Lol p 5 variant is selected from amolecule having the amino acid sequence set forth in SEQ ID NOs:8 to 12or a modified allergen corresponding to an immunologically relatedallergen.

The Lol p 5 variants identified by SEQ ID NOs:8 to 12 are referred toherein as mutants D1 to D5, respectively.

The present invention further provides a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding a modified recombinant allergen, wherein said allergenin naturally occurring form is associated with allergic diseaseconditions in sensitive subjects, wherein said modified recombinantallergen comprises an amino acid sequence modified from the naturallyoccurring amino acid sequence such that the allergen lacks or comprisesreduced numbers of IgE epitopes and/or exhibits reduced binding capacityfor IgE and/or exhibits reduced IgE production-stimulatory activitywhile retaining T-cell antigenicity.

Another aspect of the present invention provides a nucleic acid moleculecomprising a sequence of nucleotides encoding or complementary to asequence encoding a modified recombinant allergen, wherein said allergenin naturally occuring form is associated with type I allergic diseaseconditions in sensitive subjects, wherein said modified recombinantallergen comprises an amino acid sequence modified from the naturallyoccurring amino acid sequence such that the allergen lacks or comprisesreduced numbers of IgE epitopes and/or exhibits reduced binding capacityfor IgE and/or exhibits reduced IgE production-stimulatory activitywhile retaining T-cell antigenicity.

Yet another aspect of the present invention is directed to a nucleicacid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding modified recombinant grass pollenallergen, wherein said grass pollen allergen in naturally occurring formis associated with type I allergic disease conditions in sensitivesubjects wherein said modified recombinant grass pollen allergencomprises an amino acid sequence modified from the naturally occurringamino acid sequence such that the allergen lacks or comprises reducednumbers of IgE epitopes and/or exhibits reduced binding capacity for IgEand/or exhibits reduced IgE production-stimulatory activity whileretaining T-cell antigenicity.

Still yet another aspect of the present invention relates to a nucleicacid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding modified recombinant rye-grasspollen allergen comprising an amino acid sequence modified from thenaturally occurring amino acid sequence such that the allergen lacks orcomprises reduced numbers of IgE epitopes and/or exhibits reducedbinding capacity for IgE and/or exhibits reduced IgEproduction-stimulatory activity while retaining T-cell antigenicity.

Thus, a particularly preferred aspect of the present invention providespurified nucleic acid molecules encoding a modified grass pollenallergen and more particularly a modified group 5 grass pollen allergen,or an antigenic fragment thereof, or derivative or homolog thereof, orthe functional equivalent of such a nucleic acid sequence wherein themodified grass pollen allergen lacks or comprises reduced number of IgEepitopes and/or exhibites reduced binding capacity for IgE and/orexhibits reduced IgE production-stimulatory activity while retainingT-cell antigenicity. Preferred nucleic acid sequences encode group 5allergen family members such as Lol p 5, Poa p 5 and Ph1 p 5. Oneparticularly useful nucleic acid molecule encodes Lol p 5 mutants D1 toD5.

The nucleic acid molecule of the present invention may be genomic orcDNA molecules or a corresponding mRNA molecule and may be referred toas a gene. Reference to a “gene”, in respect of the present invention,means any contiguous sequence of nucleotides, the transcription of whichleads to a mRNA molecule or which sequence is a mRNA molecule, whichmRNA molecule is capable of being translated into a protein. The geneencoding a group 5 grass pollen allergen family member means thenucleotide sequence encoding the protein or a derivative or a homolog ofthe protein which may contain single or multiple amino acidsubstitutions, deletions and/or additions relative to the correspondingnaturally occurring molecule. A modified Lol p 5 gene also refers tocDNAs complementary to the mRNAs corresponding to the full or partiallength of a Lol p 5 protein having at least one truncated or amino acidsubstitution, addition and/or deletion relative to the naturallyoccuring molecules.

The present invention further contemplates fusion molecules. Forexample, for some aspects of the present invention, it is desirable toproduce a fusion protein comprising modified grass pollen allergen or afragment thereof or a derivative thereof and an amino acid sequence fromanother peptide or protein, examples of the latter being enzymes such as1-galactosidase, phosphatase, urease and the like. Most fusion proteinsare formed by the expression of a recombinant gene in which two codingsequences have been joined together such that their reading frames arein phase. Alternatively, proteins or peptides can be linked in vitro bychemical means. All such fusion protein or hybrid genetic derivatives ofa grass pollen allergen or its encoding nucleotide sequences areencompassed by the present invention. Furthermore, by homologs andderivatives of a grass pollen allergen protein is meant to includesynthetic derivatives thereof. The nucleotide sequences as elucidatedherein, can be used to chemically synthesize the entire potein orgenerate any number of fragments (peptides) by chemical synthesis bywell known methods (e.g. solid phase synthesis). All such chemicallysynthesized peptides are encompassed by the present invention.Accordingly, the present invention extends to isolated modified grasspollen allergen family members, fragments thereof and their derivatives,homologs and immunological relatives made by recombinant means or bychemical synthesis.

The terms “isolated” and “purified” are used interchangeably herein andrefer to peptides, proteins, protein fragments and nucleic acidsequences substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when synthesized chemically. The term “naturally occurring” asused herein refers to proteins or fragments thereof purified from grasspollen or other plant part. It also includes reference to an amino acidsequence determined by a cDNA sequence but which is associated withallergic conditions in a similar way to an allergen purified from grasspollen.

Fragments of nucleic acid molecules within the scope of the inventioninclude those coding for parts of grass pollen allergens that exhibitT-cell antigenicity but which lacks or exhibits reduced IgE interactionin mammals, preferably humans.

Fragments and mutants of recombinantly or synthetically producedmodified grass pollen allergens which do not bind IgE and/or which haveminimal IgE interacting ability and/or which have minimal capacity tostimulate IgE production are desirable. It is preferable that suchminimal IgE interacting activity does not lead to histamine release. Forexample, it is preferable that the modified allergen does not causecross linking of IgE on mast cells or basophils. Minimal IgE interactingactivity refers to IgE interaction activity which is less than theamount of IgE interaction by recombinantly or synthetically produced“naturally occurring” grass pollen allergen protein or whole purifiednative grass pollen allergen. IgE interaction may also be measured asIgE production stimulating activity. Preferred fragments also includeantigenic fragments which, when administered to a grass pollen-sensitiveindividual or an individual allergic to an allergen cross-reactive withgrass pollen allergen, are capable of modifying the allergic response tograss pollen allergen of the individual.

Antigenic fragments of the present invention which have T-cellstimulating activity i.e. T-cell antigenicity, and thus comprise atleast one T-cell epitope are particularly desirable. T-cell epitopes arebelieved to be involved in initiation and perpetuation of the immuneresponse to a protein allergen which is responsible for the clinicalsymptoms of allergy. These T-cell epitopes are throught to trigger earlyevents at the level of the T helper cell by binding to an appropriateHLA molecule on the surface of an antigen presenting cell andstimulating the relevant T-cell sub-population. These events lead toT-cell proliferation, lymphokine secretion, local inflammatoryreactions, recruitment of additional immune cells to the site, andactivation of the B cell cascade leading to production of antibodies.One isotype of these antibodies, IgE, is fundamentally important to thedevelopment of allergic symptoms and its production is influenced earlyin the cascade of events, at the level of the T helper cell, by thenature of the lymphokines secreted. A T-cell epitope is the basicelement or smallest unit of recognition by a T-cell receptor, where theepitope comprises amino acids essential to receptor recognition. Aminoacid sequences which mimic those of the T-cell epitopes and which modifythe allergic response to protein allergens are within the scope of thisinvention.

Exposure of patients to purified modified protein allergens of thepresent invention or to the antigenic fragments of the present inventionwhich comprise at least one T-cell epitope and are derived from proteinallergens may tolerize or anergize appropriate T-cell subpopulaions suchthat they become unresponsive to the protein allergen and do notparticipate in stimulating an immune response upon such exposure. Inaddition, administration of the protein allergen of the invention or anantigenic fragment of the present invention which comprises at least oneT-cell epitope may modify the lymphokine secretion profile as comparedwith exposure to the naturally-occurring protein allergen or portionthereof (e.g. result in a decrease of IL-4 and/or an increase in IL-2).Furthermore, exposure to such antigenic fragment or protein allergen mayinfluence T-cell subpopulations which normally participate in theresponse to the allergen such that these T-cells are drawn away from thesite(s) of normal exposure to the allergen (e.g. nasal mucosa, skin andlung) towards the site(s) of therapeutic administration of the fragmentor protein allergen. This redistribution of T-cell sub-populations mayameliorate or reduce the ability of an individual's immune system tostimulate the usual immune response at the site of normal exposure tothe allergen, resulting in a diminution in allergic symptoms.

The present invention provides expression vectors and host cellstransformed to express the nucleic acid sequences of the invention.Expression vectors of the present invention comprise a nucleic acidsequence coding for a modified grass pollen allergen, or an antigenicfragment thereof, or a derivative or homolog thereof, or the functionalequivalent of such nucleic acid sequence. The nucleic acid sequences maybe expressed in prokaryotic or eukaryotic host cells. Suitable hostcells include bacterial cells such as E. coli, insect cells, yeast, ormammalin cells such as Chinese hamster ovary cells (CHO). Suitableexpression vectors, promoters, enhancers, and other expression controlelements may be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989. Suitable vectors for expression in yeastinclude YepSec1 (Baldari et al., EMBO J 6: 229-234, 1987); pMF (Kurjanand Herskowtiz, Cell 30: 933-943, 1982); and JRY88 (Schultz et al., Gene54: 113-123, 1987).

Host cells can be transformed to express the nucleic acid sequences ofthe present invention using conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, or electroporation. Suitable methods for transforming thehost cells may be found in Sambrook et al., 1989, supra, and otherlaboratory textbooks. The nucleic acid sequences of the invention mayalso be synthesized using standard techniques.

Accordingly, another aspect of the present invention provides a methodof producing a recombinant modified grass allergen or a fragmentthereof, or a derivative or homolog thereof, or immunological relativesthereof comprising culturing an organism containing a replicablerecombinant DNA molecule, said molecule comprising a promoter capable ofexpression in said organism, a gene encoding a modified grass pollenallergen or family member, a fragment or homolog or derivative thereof,or an immunological relative thereof, located downstream of andtranscribed from said promoter, a selectable marker and a DNA vehiclecontaining a prokaryotic or eukaryotic origin of replication, underconditions and for a time sufficient for said recombinant DNA moleculeto be stably maintained and direct the synthesis of the modified grasspollen allergen or fragment or derivative, homolog or immunologicalreative thereof and then optionally isolating same.

The grass pollen allergens and fragments (peptides) thereof can bepurified from cell culture medium, host cells, or both using techniquesknown in the art for purifying peptides and proteins, includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis and immunopurification with antibodiesspecific for the modified grass pollen allergen. The terms “isolated”and “purified” are used interchangeably herein and refer to peptides,proteins, protein fragments, and nucleic acid sequences substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whensynthesized chemically.

Another aspect of the invention provides protein preparations comprisingLol p5 D1, D2, D3, D4 and D5 or their functional or immunologicalequivalents, homologs or derivatives.

Thus, the present invention provides modified grass pollen allergens ortheir derivatives which, when administered to a grass pollen-sensitiveindividual, reduce the allergic response of the individual to grasspollen such as rye-grass pollen or pollen from immunologically relatedgrasses. Preferred modified grass pollen allergens include modified Lolp5 protein or a derivative or homolog thereof. Other preferred allergensare Ph1 p 5 and Poa p 5.

In addition to inducing an amino substitution, addition and/or deletionor truncation, another example of a modification of proteins or peptidesis substitution of cysteine residues preferably with alanine, serine,threonine, leucine or glutamic acid to minimize dimerization viadisulfide linkages. Another example of modification of the proteins andpeptides of the invention is by chemical modification of amino acid sidechains or cyclization of the peptide.

In order to enhance stability and/or reactivity, proteins or peptides ofthe invention can also be modified to incorporate one or morepolymorphisms in the amino acid sequence of the protein allergenresulting from natural allelic variation. Additionally, D-amino acids,non-natural amino acids or non-amino acid analogs can be substituted oradded to produce a modified protein or peptide within the scope of thisinvention.

Another aspect of the present invention relates to recombinant vectorscomprising DNA sequences encoding proteins displaying modifiedallergenic activity from pollen of a grass species. More particularly,the grass species belongs to the family Poaceae (Gramineae), and evenmore particularly, to the genus Lolium. Still even more particularly,the allergenic protein is characterized as being immunologicallycross-reactive with antibody to Lol pIb protein of Lolium perennepollen, namely:

-   -   Pooid (festucoid) grasses. Group 1: Triticanea: Bromus inermis,        smooth broom; Agropyron repens, English couch; A. cristatum;        Secale cereale rye Triticum aestivum wheat. Group 2: Poanae:        Dactylis glomerata, orchard grass of perennial ryegrass; L.        multiflorum, Italian ryegrass; Poa pratensis, Kentucky        bluegrass; P. compressa, flattened meadow grass; Avena sativa,        oat; Holcus lanatus, velvet grass or Yorkshire fog; Anthoxanthum        odoratum; sweet vernal grass; Arrhenatherum elatius, oat grass;        Agrostis alba, red top; Phleum pratense, timothy; Phalaris        arundinacea, reed canary grass. Panicoid grass, Paspalum        notatum, Bahia grass, Andropogonoid grasses: Sorghum halepensis,        Johnson grass.

A variety of expression vectors can be constructed for the production ofa modified grass pollen allergen or a fragment or derivative thereof.

The present invention extends to monoclonal and polyclonal antibodies tomodified grass pollen allergens or fragments, derivatives or homologsthereof.

The monoclonal antibodies are useful to screen cDNA libraries or topurified recombinantly produced proteins or even in therapy to reducethe activity of an introduced protein. In the following discussion,reference to grass pollen protein allergens include their derivatives,homologs and immunological relatives and chemical synthetic derivativesthereof. The following discussion also includes antibodies specific forpurified modified Lol p 5 and fragments, derivatives and homologsthereof. Such antibodies are contemplated to be useful in developingdetection assays (immunoassays) for modified grass pollen allergensespecially during the monitoring of a therapeutic or diagnostic regimenand in the purification of recombinantly or synthetically produced grasspollen family members and in particular group 5 grass pollen allergen.The antibodies may be monoclonal or polyclonal. Additionally, it iswithin the scope of this invention to include any second antibodies(monoclonal or polyclonal) directed to the first antibodies discussedabove. The present invention further contemplates use of these first orsecond antibodies in detection assays and, for example, in monitoringthe effect of a diagnostic or an administered pharmaceuticalpreparation. Furthermore, it is within the scope of the presentinvention to include antibodies to any molecules complexed with amodified grass pollen protein allergen. Accordingly, an antibody to agrass pollen protein allergen encompasses antibodies to such a proteinallergen, or antigenic parts thereof, and to any associated molecules(e.g. lipid regions, carrier molecules, fused proteins, and the like).

The grass pollen family members, or fragments thereof, considered hereinare purified then utilized in antibody production. Both polyclonal andmonoclonal antibodies are obtainable by immunization with recombinant orsynthetic modified grass pollen protein family members, and either typeis utilizable for immunoassays. The methods of obtaining both types ofsera are well known in the art. Polyclonal sera are less preferred butare relatively easily prepared by injection of a suitable laboratoryanimal with an effective amount of a purified modified grass pollenallergen, or antigenic parts thereof, collecting serum from the animal,and isolating specific sera by any of the known immunoabsorbenttechniques. Although antibodies produced by this method are utilizablein virtually any type of immunoassay, they are generally less favoredbecause of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example, Kohler and Milstein, Nature 256: 495-499,1975; Kohler and Milsten, Eur. J. Immunol 6: 511-519, 1976).

Unlike preparation of polyclonal sera, the choice of animal is dependenton the availability of appropriate immortal lines capable of fusing withlymphocytes. Mouse and rat have been the animals of choice in hybridomatechnology and are preferably used. Humans can also be utilized assources for sensitized lymphocytes if appropriate immortalized human (ornon-human) cell lines are available. For the purpose of the presentinvention, the animal of choice may be injected with from about 0.1 mgto about 20 mg of purified modified grass pollen allergen or partsthereof. Usually the injecting material is emulsified in Freund'scomplete adjuvant. Boosting injections may also be required. Thedetection of antibody production can be carried out by testing theantisera with appropriately labeled antigen. Lymphocytes can be obtainedby removing the spleen or lymph nodes of sensitized animals in a sterilefashion and carrying out fusion. Alternatively, lymphocytes can bestimulated or immunized in vitro.

The presence of modified grass pollen allergens contemplated herein, orantibodies specific for same, in a patient's serum, plant or mammaliantissue or tissue extract, can be detected utilizing antibodies preparedas above, either monoclonal or polyclonal, in virtually any type ofimmunoassay. A wide range of immunoassay techniques are available as canbe seen by reference to U.S. Pat. Nos. 4,015,043, 4,424,279 and4,018,653. This includes both single-site and two-site, or “sandwich”,assays of the non-competitive types, as well as in the traditionalcompetitive binding assays. Sandwich assays are among the most usefuland commonly used assays and are favored for use in the presentinvention. A number of variations of the sandwich assay technique exist,and all are intended to be encompassed by the present invention.Briefly, in a typical forward assay, an unlabeled antibody isimmobilized in a solid substrate and the sample to be tested broughtinto contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen secondary complex, a second antibody, labeled with areporter molecule capable of producing a detectable signal is then addedand incubated, allowing time sufficient for the formation of a tertiarycomplex of antibody-antigen-labeled antibody (e.g., antibody-modifiedgrass pollen allergen protein-antibody). Any unreacted material iswashed away, and the presence of the antigen is determined byobservation of a signal produced by the reporter molecule. The resultsmay either be qualitative, by simple observation of the visible signal,or may be quantitated by comparing with a control sample containingknown amounts of hapten. Variations on the forward assay include asimultaneous assay, in which both sample and labeled antibody are addedsimultaneously to the bound antibody, or a reverse assay in which thelabeled antibody and sample to be tested are first combined, incubatedand then added simultaneously to the bound antibody. These techniquesare well known to those skilled in the art, including any minorvariations as will be readily apparent.

Although the following discussion is concerned with detecting modifiedgrass pollen allergen, it is equally applicable to detecting antibodiesto same and it is intended to be a sufficient description thereof.

In the typical forward sandwich assay a first antibody havingspecificity for modified grass pollen allergen, or antigenic partsthereof, contemplated in this invention, is either covalently orpassively bound to a solid surface. The solid surface is typically glassor a polymer, the most commonly used polymers being cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.The solid supports may be in the form of tubes, beads, discs ofmicroplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated from about room temperature to about37° C. for a period of time sufficient to allow binding of any subunitpresent in the antibody. The incubation period will vary but willgenerally be in the range of about 2-40 minutes or overnight if moreconvenient. Following the incubation period, the antibody subunit solidphase is washed and dried and incubated with a second antibody specificfor a portion of the hapten. The second antibody is linked to a reportermolecule which is used to indicate the binding of the second antibody tothe hapten.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes). In the case of an enzyme immunoassay, an enzyme isconjugated to the second antibody, generally by means of glutaraldehydeor periodate. As will be readily recognized, however, a wide variety ofdifferent conjugation techniques exist, which are readily available tothe skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, β-galactosidase and alkaline phosphatase,amongst others. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates, for peroxidase conjugates, 1,2-phenylenediamine,5-aminosalicylic acid, or toluidine are commonly used. It is alsopossible to employ fluorogenic substrates, which yield a fluorescentproduct rather than the chromogenic substrates noted above. In allcases, the enzyme-labeled antibody is added to the first antibody haptencomplex, allowed to bind, and then the excess reagent is washed away. Asolution containing the apropriate substrate is then added to thetertiary complex of antibody-antigen-antibody. The substrate will reactwith the enzyme linked to the second antibody, giving a qualitativevisual signal, which may be further quantitated, usuallyspectophotometrically, to give an indication of the amount of haptenwhich was present in the sample. “Reporter molecule” also extends to useof cell agglutination or inhibition of agglutination such as red bloodcells or latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labeled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength, the fluorescein observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemilluminescent or bioluminescentmolecules, may also be employed. It will be readily apparent to theskilled technician how to vary the procedure to suit the requiredpurpose. It will also be apparent that the foregoing can be used todetect directly or indirectly (i.e. via antibodies) grass pollenallergen protein of this invention.

Accordingly, one aspect of the present invention provides a method ofdetecting a modified pollen allergen or a derivative or homolog thereofor an allergenic protein immunologically reactive with said modifiedgrass pollen allergen or a derivative or homolog present in serum,tissue extract, plant extract or other biological fluid or compositioncomprising the steps of containing said fluid or composition to betested with an antibody to said modified grass pollen protein allergenfor a time and under conditions sufficient for a modified allergenicprotein-antibody complex to form and subjecting said complex to adetecting means. For purification methods, an antibody to a nativeallergen may also be effective in order to purify a modified allergenand such an embodiment is encompassed by the present invention.

The present invention is also directed to a kit for the rapid andconvenient assay for antibodies to modified grass pollen allergens orderivatives, homologs or immunological relatives thereof in mammalianbody fluids e.g. serum, tissue extracts, tissue fluids), in vitro cellculture supernatants, and cell lysates. The kit is compartmentalized toreceive a first container adapted to contain an antigenic componentthereof, and a second container adapted to contain an antibody to thegrass pollen allergen, said antibody being labeled with a reportermolecule capable of giving a detectable signal. If the reporter moleculeis an enzyme, then a third container adapted to contain a substrate forsaid enzyme is provided. In an exemplified use of the subject kit, asample to be tested is contacted with the contents of the firstcontainer for a time and under conditions for an antibody, if present inthe sample, to bind to the grass pollen allergen in said firstcontainer.

Because of the presence of allergens in the environment, hayfever andseasonal asthma continue to have significant morbidity andsocio-economic impact on Western communities, despite advances made intheir pharmacology and immunology. While the available spectrum ofdrugs, including antihistamines and steroids have resulted inimprovement in the treatment of allergic disease, they have unfortunateside-effects associated with long term usage. Because of these problems,renewed interest has been shown in the immunotherapy of allergicdisease. Immunotherapy involves the injection of potent allergenextracts to desensitize patients against allergic reactions.Unfortunately, the pollen preparations used as allergens are polyvalentand of poor quality. Consequently, concentrations used are frequentlyhigh in order to induce IgG responses, but may be lethal throughtriggering of systemic reactions, including anaphylaxis. The cloned geneproduct or synthetic peptides based on the sequence of allergensprovides a safer medium for Therapy since it can be quality controlled,characterized and standardized.

Accordingly, the present invention contemplates a method fordesensitizing a mammal (e.g. human) allergic to grass pollen whichcomprises administering to said mammal a desensitizing-effective amountof a modified grass pollen allergen which lacks or comprises reducednumbers of and/or exhibits reduced IgE binding activity and/or exhibitsreduced IgE production-stimulatory activity or a fragment or derivative,homolog, or immunological relative thereof, for a time and underconditions sufficient to effect desensitization of the mammal (e.g.human) to the grass pollen.

The present invention also provides a method of treating sensitivity toryegrass pollen or pollen from an immunological relative of rye-grass ina mammal (e.g. human) sensitive to such pollen, comprising administeringto the mammal a therapeutically effective amount of a therapeuticcomposition of the invention. The present invention further provides amethod of treating sensitivity to ryegrass pollen allergen or anallergen immunologically cross-reactive with ryegrass pollen allergencomprising administering to a mammal a therapeutically effective amountof said protein preparation of the invention.

Through the use of the peptides and protein of the present invention,preparations of consistent, well-defined composition and biologicalactivity can be made and administered for therapeutic purposes (e.g. tomodify the allergic response of a L. perenne sensitive individual topollen of such plant. Administration of such peptides or protein may,for example, modify IgE response to the grass pollen allergen. Purifiedpeptides can also be used to study the mechanism of immunotherapy of L.perenne allergy and to design modified derivatives or analogs useful inimmunotherapy.

The present invention is directed, therefore, to the use of a modifiedallergen in the manufacture of a medicament for the treatment orprophylaxis of allergen-sensitive individuals.

The present invention, therefore, provides a pharmaceutical compositioncomprising a desensitizing or therapeutically effective amount ofmodified grass pollen allergen and in particular group 5 grass pollenallergen or derivatives, homologs or immunological relatives thereof andone or more pharmaceutically acceptable carriers and/or diluents. Theactive ingredients of a pharmaceutical composition comprising themodified grass pollen allergen prophylactic is contemplated to exhibitexcellent therapeutic or activity, for example, in the desensitizationof humans allergic to grass pollen when administered in amount whichdepends on the particular case. For example, from about 0.5 Fg to about20 mg per kilogram of body weight per day may be administered. Dosageregime may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation. The activity compound may be administered in aconvenient manner such as by the oral, intravenous (where watersoluble), intramuscular, subsutaneous, intranasal, intradermal orsuppository routes or implanting (e.g. using slow release molecules).Depending on the route of administration, the active ingredients whichcomprise the pharmaceutical composition of the invention may be requiredto be coated in a material to protect said ingredients from the actionof enzymes, acids and other natural conditions which may inactivate saidingredients. For example, the modified grass pollen allergen may beadministered in an adjuvant, co-administered with enzyme inhibitors orin liposomes. Adjuvant is used in its broadest sense and includes anyimmune stimulating compound, such as interferon. Adjuvants contemplatedherein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin. Liposomes includewater-in-oil-in-water CF emulsions as well as conventional liposomes.For purposes of inducing T cell anergy, the pharmaceutical compositionif preferably administered in non-immunogenic form (e.g. it does notcontain adjuvant).

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions or sterilepowders for preparation of injectable solutions. In all cases the formmust be sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, iropylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of superfactants. Thepreventions of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When a modified grass pollen allergen, or a fragment thereof is suitablyprotected as described above, the active compound may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets or it may be incorporateddirectly with food of the diet. For oral therapeutic administration, theactive compound may be incorporated with excipients and used in the formof ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such cmpositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, becarried and may conveniently be between about 5 to 80% of the weight ofthe unit. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention areprepared so that an oral dosage unit form contains between about 10 Fgand 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain theingredients listed below: A binder such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and prppylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Generation of Lol p 5 Mutant Proteins

To engineer hypoallergenic, non-IgE reactive allergen variants, it wasrequired to be determined whether key residues of the proteins can beselected which can be changed while keeping the general structure andT-cell epitopes intact. Since the highest frequency of IgE binding isobserved in peptide fragments which span the C-terminal half of Lol p 5,the inventors introduced mutations predominantly in the C-terminus ofthe allergen. To identify amino acid positions in Lol p 5 likely to havean influence on the IgE intereactivity of the protein, protein sequencesof isoform A and B of Lol p 5 were compared with group 5 allergens ofother grasses (FIG. 1). Site-directed mutagenesis was employed toreplace residues that are highly conserved among group 5 allergens (FIG.2). Mutant proteins altered in one or three domains were generated (FIG.3).

Non-mutated Lol p 5 and mutated variants of Lol p 5 were expressed insoluble forms in E. coli using the pQE expression vector system(Qiagen). Bacterial expression (see below) of proteins using this vectorintroduces a polyhistidine tag at the N-terminus of the molecules whichis useful for purification of recombinant proteins by one step metalchelate affinity chromatography. Non-mutagenized control and mutatedproteins were then tested for IgE reactivity as well as for reactivitywith anti-Lol p 5 monoclonal antibody A7 (Mab A7) and polyclonalanti-Lol p 5 antiserum in slot blots (FIG. 5), Western blots (FIG. 6)and ELISA assays (FIG. 7). The results showed a substential reduction inIgE binding activity in case of several of the mutated proteins (e.g.mut 4, mut 6, mut 9). Such engineered allergenic molecules arepotentially useful for safer and more effective immunotherapy for type Iallergic diseases and the described approach may be generally applied toproduce non-IgE reactive variants of allergens.

EXAMPLE 2 Expression and Purification of Recombinant Lol P 5 and MutantProteins

The coding sequences of Lol p 5 and the mutant proteins were introducedin-frame into the expression vector pQE31 (QIAGEN). The vector allowsexpression of recombinant proteins with an N-terminal, 6-residuehistidine tag. Expression and harvesting of the proteins was carried outas outlined in the QIA expressionist manual. Histidine-tagged proteinswere purified using TALON metal affinity resin (Clontech), following theprocedure for batch/gravity flow column purification as outlined in theTALON Metal Affinity Resin user manual (Clontech).

EXAMPLE 3 SDS-Page and Western Blotting

For SDS-PAGE, 1.3 Fg of Lol p5 and each of the mutant proteins wereboiled for 5 minutes with 10× protein sample buffer. Samples were loadedonto a 15% w/v acrylamide mini gel at 200 V for 40 minutes in a bufferof 0.2 M glycine, 0.025 M Tris, 0.1% w/v SDS.

For staining, gels were shaken in 0.1% w/v Coomassie Brilliant Blue R250for at least an hour. Gels were destained in 20% v/v methanol, 7% v/vglacial acetic acid, 3% v/v glycerol, overnight with two buffer changes.

Gels were western blotted in a BIORAD mini-Protean II cell western blotapparatus in a buffer of 0.025 M Tris, 0.2 M glycine, 20% v/v methanolonto Nytran 0.2 Fm nylon membrane (Schleicher & Schuell) at 100 V for 1hour at 4° C.

EXAMPLE 4 Slot Blot Analysis

For slot blot analysis, 0.7 Fg of mutant proteins and Lol p5 were addedinto the slots of a Hybri-Slot manifold slot blot apparatus (LifeTechnologies, Inc.) and blotted onto Nytran 0.2 Fm nylon membrane(Schleicher & Schuell) under suction from a water vacuum.

EXAMPLE 5 Incubation of Blots with Antibodies and Patient Sera

Prior to incubation with antibodies or sera, all western and slot blotswere blocked in 10% w/v skim milk powder in PBS (150 mM sodium chloride,36 mM sodium phosphate monobasic, monohydrate, 7 mM sodium phosphatedibasic, dihydrate) for one hour with shaking. Blots were washed oncewith PBS, 0.5% v/v Tween 20, twice with PBS and incubated overnight withmonoclonal antibodies (mAb A7: diluted 1:5), polyclonal antibodies (B1:diluted 1:50) or patient sera. All dilutions were prepared in PBS, 0.5%w/v BSA, 0.1% w/v sodium azide, and shaken with the blots overnight atroom temperature. After washing as above blots were incubated withalkaline-phosphatase conjugated anti-mouse (mAb A7) or anti-rabbit (B1)secondary antibodies (Promega) diluted 1:5000 in PBS, 0.5% v/v Tween 20,1% w/v BSA for 1 hour with shaking at room temperature. All blots werethan washed as above. Bound anti-mouse and anti-rabbit antibodies weredetected by a colour reaction—10 ml alkaline phosphatase buffer (0.1 MTris, pH 9.5, 0.1 M sodium chloride, 0.05 M magnesium chloride) with 66Flof BCIP stock (5% w/v bromochloroindolyl phosphate in 100% v/vdimethylfomamide). Blots incubated with patient sera were probed withI¹²⁵-labeled anti-human antibody (Bioclone) diluted 1:5 in PBS, 0.5% v/vTween 20, 1% w/v BSA (buffer B) overnight with shaking at roomtemperature. All blots were washed as above. After washing, bound125-labeled anti-human IgE was detectd by exposure to Kodak Biomax MSfilm at −70° C.

EXAMPLE 6 Direct ELISA

The wells of an ELISA plate (Greiner) were coated with 50 Fl aliquots of100, 500, 1000, 5000 and 10000 ng/ml dilutions of Lol p5 and the fourmutant proteins, and incubated at 4° C. overnight. The wells were thenwashed four times with PBS, 0.5% v/v Tween 20. After blocking withbuffer B at room temperature for one hour, the wells were washed again,and incubated with 50 Fl of an appropriate dilution of patient sera inbuffer B at 4° C. overnight. Wells were washed as above beforeincubation with 50 Fl of a 1:2000 dilution of anti-human IgE antibody(Alkaline-Phosphatase conjugated: Sigma) in buffer B at room temperaturefor one hour. After a final series of washes, bound anti-human IgE wasdetected with Blue Phos microwell phosphatase substrate system(Kirkegaard & Perry Laboratories). Colour development was detected by aSpectracount plate reader at 630 m (Packard).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

-   Bond, J. F, Segal, D. B, Yu X-B, Theriault, K. A, Pollock, M. S,    Yeung H. J. Allergy Clin. Immunol. 91: 339, 1993.-   Baldari et al, EMBO J. 6: 229-234, 1989.-   Kohler and Milstein, Nature 256: 495-499, 1975.-   Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976.-   Kurjan and Herskowitz, Cell 30: 933-943, 1982.-   Miyamoto T: Advances in Allergology and Clinical Immunology. Godard    P, Bousquet J, Michel FB (eds) pp. 343-347. The Parthenon Publishing    Group, Cornforth, UK, 1992.-   Ong, E. K, Griffith, I. J., Knox, R. B., Singh, M. B. Gene 134:    235-240, 1993.-   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.    Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.-   Schultz et al, Gene 54: 113-123, 1987.-   Singh, M. B., Hough, T., Theerakulpisut, P., Avjioglu, A., Davies,    S., Smith, P. M., Taylor, P., Simpson, R. J., Ward. L. D.,    McCluskey, J., Puy, R., Knox, R. B. Proc. Natl. Acad. Sci USA 88:    13841388, 1991.-   Smart, I. J., Heddle, R. J., Zola, H., Bradley, J., Int. Arch.    Allergy Immunol. 72: 243-248, 1983.-   Smith, P. M., Ong, E. K, Avjioglu, A., Singh, M. B., Knox, R. B.    Analysis of rye-grass pollen allergens using two dimensional    electrophoresis and immunoblotting. In Kraft D (ed), Molecular    Biology and Immunology of Allergens, CRC Press, Boca Raton, Fla.,    1993.-   Smith, P. M., Ong, E. K., Knox, R. B., Singh, M. B. Mol. Immunol.    31: 491-498, 1994.-   Wuthrich, B., Int. Arch. Allergy Immunol. 90: 3-10, 1989.

1. A modified recombinant allergen, wherein said allergen in naturallyoccurring form is associated with allergic disease conditions insensitive subjects, wherein said modified recombinant allergen comprisesan amino acid sequence modified from the naturally occurring amino acidsequence such that the allergen lacks or comprises reduced numbers ofIgE epitopes and/or exhibits reduced binding capacity for IgE and/orexhibits reduced IgE production stimulatory activity while retainingT-cell antigenicity.
 2. The modified recombinant allergen of claim 1,wherein said allergen is a grass pollen allergen.
 3. The modifiedrecombinant allergen of claim 2, wherein said grass pollen allergencomprises a substitution, deletion and/or addition at one or more aminoacid residues which is/are conserved in at least two immunologicallycross-reactive group 5 grass pollen allergens and wherein said modifiedgroup 5 grass pollen allergen lacks or comprises reduced numbers of IgEepitopes and/or exhibits reduced binding capacity for IgE and/orexhibits reduced IgE production stimulatory activity compared to thecorresponding naturally occurring form.
 4. The modified recombinantallergen of claim 3, wherein said grass pollen allergen is a rye grasspollen allergen.
 5. The modified recombinant allergen of claim 2 or 3 or4, wherein said allergen is selected from the list comprising Lol p 5,Ph1 p 5, Pao p 5 and immunologically-related allergens.
 6. The modifiedrecombinant allergen of claim 5, wherein said allergen comprises anamino acid truncation or substitution, deletion and/or addition at aposition corresponding to one or more of mutants 1 to 9 of Lol p 5 or acorresponding mutant in an immunologically related allergen.
 7. Themodified recombinant allergen of claim 6, wherein said allergencomprises a Lol p 5 variant comprising a molecule having the amino acidsequence set forth in any one of SEQ ID NO:8 to SEQ ID NO:12 or amodified allergen corresponding to an immunologically related allergen.8. The modified recombinant allergen of claim 1, wherein the allergicdisease condition is a type I allergic disease.
 9. The modifiedrecombinant allergen of claim 8, wherein the type I allergic disease issensitivity to rye grass pollen.
 10. The modified recombinant allergenof claim 1, wherein said sensitive subject is a human, a primate,livestock animal, laboratory test animal or companion animal.
 11. Themodified recombinant allergen of claim 10, wherein said sensitivesubject is a human.
 12. A composition comprising a modified allergenwhich lacks or comprises reduced numbers of IgE epitopes and/or exhibitsreduced binding capacity for IgE and/or exhibits reduced IgE productionstimulatory activity while retaining T-cell antigenicity and one or morepharmaceutically acceptable carriers and/or diluents.
 13. Thecomposition of claim 12, wherein said modified allergen is a grasspollen allergen.
 14. The composition of claim 13, wherein said grasspollen allergen comprises a substitution, deletion and/or addition atone or more amino acid residues which is/are conserved in at least twoimmunologically cross-reactive group 5 grass pollen allergens andwherein said modified group 5 grass pollen allergen lacks or comprisesreduced numbers of IgE epitopes and/or exhibits reduced binding capacityfor IgE and/or exhibits reduced IgE production stimulatory activitycompared to the corresponding naturally occurring form.
 15. Thecomposition of claim 14, wherein said grass pollen allergen is a ryegrass pollen allergen.
 16. The composition of claim 15, wherein saidallergen is selected from the list comprising Lol p 5, Ph1 p 5, Pao p 5and immunologically-related allergens.
 17. The composition of claim 16,wherein said allergen comprises an amino acid truncation orsubstitution, deletion and/or addition at a position corresponding toone or more of mutants 1 to 9 of Lol p 5 or a corresponding mutant in animmunologically related allergen.
 18. The composition of claim 17,wherein said allergen comprises a Lol p 5 variant comprising a moleculehaving the amino acid sequence set forth in any one of SEQ ID NO:8 toSEQ ID NO:12 or a modified allergen corresponding to an immunologicallyrelated allergen.
 19. A method for the prophylaxis or treatment of anallergic disease condition in a subject, said method comprisingadministering to said subject an effective amount of a modified allergenwhich lacks or comprises reduced numbers of IgE epitopes and/or exhibitsreduced binding capacity for IgE and/or exhibits reduced IgE productionstimulatory activity while retaining T-cell antigenicity.
 20. The methodof claim 19, wherein said allergen is a grass pollen allergen.
 21. Themethod of claim 20, wherein said grass pollen allergen comprises asubstitution, deletion and/or addition at one or more amino acidresidues which is/are conserved in at least two immunologicallycross-reactive group 5 grass pollen allergens and wherein said modifiedgroup 5 grass pollen allergen lacks or comprises reduced numbers of IgEepitopes and/or exhibits reduced binding capacity for IgE and/orexhibits reduced IgE production stimulatory activity compared to thecorresponding naturally occurring form.
 22. The method of claim 21,wherein said grass pollen allergen is a rye grass pollen allergen. 23.The method of claim 22, wherein said allergen is selected from the listcomprising Lol p 5, Ph1 p 5, Pao p 5 and immunologically-relatedallergens.
 24. The method of claim 23, wherein said allergen comprisesan amino acid truncation or substitution, deletion and/or addition at aposition corresponding to one or more of mutants 1 to 9 of Lol p 5 or acorresponding mutant in an immunologically related allergen.
 25. Themethod of claim 24, wherein said allergen comprises a Lol p 5 variantcomprising a molecule having the amino acid sequence set forth in anyone of SEQ ID NO:8 to SEQ ID NO:12 or a modified allergen correspondingto an immunologically related allergen.
 26. The method of claim 19,wherein the allergic disease condition is a type I allergic disease. 27.The method of claim 26, wherein the type I allergic disease issensitivity to rye grass pollen.
 28. The method of any one of claim 19,wherein said subject is a human, a primate, livestock animal, laboratorytest animal or companion animal.
 29. The method of claim 21, whereinsaid subject is a human.